The long term goal of the proposed research is to understand the role of the Drosophila SIR2 protein in gene silencing and investigate its possible role in lifespan-extension by caloric restriction (CR) in higher eukaryotes. In the budding yeast Saccharomyces cerevisiae, SIR2 has been demonstrated to be a determinant of lifespan, a single extra copy of the Sir2 gene extending lifespan by up to 50 percent. CR promotes a similar extension of yeast lifespan that is dependent on SIR2. It is of great interest to discover whether the SIR2 orthologs in higher eukaryotes are also involved in determining lifespan and lifespan extension by CR. We have identified the Drosophila SIR2 ortholog (dSIR2) by database searches and have identified mutations affecting it. We plan to use these mutations, as well as alterations in SIR2 gene dosage and directed overexpression of dSIR2 to investigate its role in gene silencing and longevity. We will characterize the biochemical activities of dSIR2, in particular determining whether it has an NAD+-dependent histone deacetylase activity that is conserved in yeast and mouse SIR2 proteins. We also plan to carry out biochemical purification of complexes containing dSIR2 by conventional chromatography and identify their polypeptide components by peptide sequencing. In preliminary studies we have made intriguing connections between dSIR2 and Polycomb Group proteins, which are involved in stable long-term silencing of the homeotic genes in Drosophila and are highly conserved in higher eukaryotes. We plan to explore this connection further through genetic and biochemical studies to determine its functional significance. We expect that these studies will provide new insights into the role of dSIR2 in gene silencing and reveal whether and how it plays a role in determining lifespan and lifespan extension by CR in higher eukaryotes.